Do you know any procedure to reduce the amount of haemoglobin which is co-purified with the protein of interest during cobalt affinity chromatography? We've just started to use the Promega reticulocyte lysate kit as a cell free protein expression system. After translation we have both the protein of interest (13 kDa) and haemoglobin. Unfortunatelly, after purification, we got mostly haemoglobin, instead of the protein, we were expecting. Promega's advice is to use their MagZ™ Protein Purification System, however I would like to avoid it. Any ideas?
There should be differences in binding affinities of these proteins to the Co column. Can you do a linear (or step wise) imidazole gradient?
I also worked with the reticulocyte lysate a while ago. First of of we did a qick spin of the lysate before using the supernatant only.
To get rid of hemoglobin we did an ammonium sulfate precipitation.
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